U.S. patent application number 15/879963 was filed with the patent office on 2018-08-02 for methods for detecting anti-he4 antibodies and methods of diagnosis and/or prognosis of conditions associated with he4-expressing cells.
This patent application is currently assigned to University of Washington through its Center for Commercialization. The applicant listed for this patent is University of Washington through its Center for Commercialization. Invention is credited to Ingegerd Hellstrom, Karl Erik Hellstrom, Jade Jaffar, Pu Liu, Elizabeth Swisher.
Application Number | 20180217158 15/879963 |
Document ID | / |
Family ID | 45724080 |
Filed Date | 2018-08-02 |
United States Patent
Application |
20180217158 |
Kind Code |
A1 |
Hellstrom; Karl Erik ; et
al. |
August 2, 2018 |
METHODS FOR DETECTING ANTI-HE4 ANTIBODIES AND METHODS OF DIAGNOSIS
AND/OR PROGNOSIS OF CONDITIONS ASSOCIATED WITH HE4-EXPRESSING
CELLS
Abstract
The present invention features, inter alia, compositions and
methods related to the detection of HE4-expressing tumor cells in a
subject. The methods include detection of anti-HE4 antibodies in a
biological sample obtained from the subject. The methods are useful
for diagnosis and monitoring the efficacy of treatments for cancers
in which HE4 is expressed, for example, ovarian cancer.
Inventors: |
Hellstrom; Karl Erik;
(Seattle, WA) ; Hellstrom; Ingegerd; (Seattle,
WA) ; Liu; Pu; (Rockville, MD) ; Jaffar;
Jade; (Camperdown, AU) ; Swisher; Elizabeth;
(Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Washington through its Center for
Commercialization |
Seattle |
WA |
US |
|
|
Assignee: |
University of Washington through
its Center for Commercialization
Seattle
WA
|
Family ID: |
45724080 |
Appl. No.: |
15/879963 |
Filed: |
January 25, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13818466 |
May 13, 2013 |
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PCT/US2011/049274 |
Aug 26, 2011 |
|
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15879963 |
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61377387 |
Aug 26, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/6854 20130101;
G01N 2800/52 20130101; G01N 33/57449 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; G01N 33/574 20060101 G01N033/574 |
Claims
1. A method for detecting the presence of HE4-expressing cells in a
human subject comprising determining the presence or amount of
anti-HE4 antibodies in a biological sample obtained from the human
subject, wherein the presence or amount of anti-HE4 antibodies in
the biological sample is indicative of the presence of
HE4-expressing cells in the human subject.
2. The method of claim 1, wherein the presence or amount of
anti-HE4 antibodies in the biological sample is determined by
contacting the biological sample with a polypeptide encoded by a
polynucleotide that selectively hybridizes to a sequence at least
90% identical to a sequence comprising at least 20 contiguous
nucleotides of SEQ ID NO:1.
3. The method of claim 1, further comprising comparing the
determined amount of anti-HE4 antibodies to a reference standard,
wherein an amount of anti-HE4 antibody detected that is greater
than the reference standard is indicative of the presence of
HE4-expressing cells in the human subject.
4. The method of claim 1, further comprising performing at least
one additional diagnostic assay to determine if the subject with
anti-HE4 antibodies has ovarian cancer.
5. The method of claim 1, wherein binding of the polypeptide to an
anti-HE4 antibody comprises detecting a signal selected from the
group consisting of a radionuclide, a fluorophore, a binding event
between an avidin molecule and a biotin molecule, a binding event
between a streptavidin molecule and a biotin molecule, and a
product of an enzyme reaction.
6. The method of claim 2, wherein the amount of anti-HE4 antibodies
in the biological sample is determined using an ELISA assay.
7. The method of claim 3, wherein the presence of HE4-expressing
cells in a human subject is indicative that the subject is
suffering from, or is at risk for, developing ovarian cancer.
8. The method of claim 1, wherein the biological sample is a
biological fluid selected from the group consisting of blood,
plasma, serum, ascitic fluid, urine, saliva, tears, pleural fluid,
sputum, vaginal fluid, and washings obtained during a medical
procedure.
9. The method of claim 1 wherein the human subject is undergoing
therapeutic treatment for a cancer associated with HE4-expressing
tumor cells.
10. A method for monitoring the efficacy of treatment of a human
cancer patient undergoing therapeutic treatment for an
HE4-expressing tumor, the method comprising: (a) providing a
biological sample from a human patient undergoing therapeutic
treatment for a cancer associated with HE4-expressing tumor cells;
(b) determining the presence or amount of anti-HE4 antibodies in
the biological sample by contacting the biological sample with a
polypeptide encoded by a polynucleotide that selectively hybridizes
to a sequence at least 90% identical to a sequence comprising at
least 20 contiguous nucleotides of SEQ ID NO:1; and (c) comparing
the determined presence or amount of anti-HE4 antibodies to an
antibody reference standard, wherein an amount of anti-HE4 antibody
greater than the reference standard is indicative of a positive
response to the therapeutic treatment for the cancer.
11. The method of claim 10, wherein the reference standard is
determined from a biological sample obtained from healthy control
subjects.
12. The method of claim 10, wherein the reference standard is
determined from a biological sample obtained from the patient prior
to treatment for cancer.
13. The method of claim 10, wherein the antibody specifically binds
to a polypeptide comprising a sequence at least 95% identical to a
sequence comprising at least 10 contiguous amino acids of SEQ ID
NO:2.
14. The method of claim 10, wherein the biological sample is a
biological fluid selected from the group consisting of blood,
plasma, serum, ascitic fluid, urine, saliva, tears, pleural fluid,
sputum, vaginal fluid, and washings obtained during a medical
procedure.
15. The method of claim 10, wherein the amount of anti-HE4
antibodies is determined using an ELISA assay.
16. The method of claim 1, further comprising determining the
presence or amount of soluble HE4-related peptides (SHRP) in the
biological sample, wherein the presence or amount of anti-HE4
antibodies in the biological sample in combination with the
presence or amount of soluble HE4-related peptides is indicative of
the presence of HE4-expressing cells in the human subject.
17. The method of claim 1 or 10, wherein the biological sample is
serum.
18. The method of claim 10, wherein the treatment includes
administration of at least one of a chemotherapeutic agent,
radiation treatment, antibody therapy, cancer vaccine therapy, gene
therapy, or stem cell transplant.
19. The method of claim 10, wherein the treatment includes surgery
to remove at least a portion of a tumor expressing HE4.
20. A kit for detecting the presence of HE4-expressing cells in a
human subject, the kit comprising reagents specific for detection
of the presence or amount of anti-HE4 antibodies in a biological
sample obtained from a human subject and printed instructions for
comparison of the detected presence or amount of anti-HE4
antibodies with a reference standard.
21. The kit of claim 20, wherein the reference standard is selected
from the group consisting of a specific numerical threshold, a
negative control sample for concurrent evaluation, and statistical
information correlating the amount of anti-HE4 antibodies detected
with the likelihood of the presence of HE4-expressing cells in the
subject.
22. The kit of claim 20, wherein the reagents specific for
detection of anti-HE4 antibodies comprise a polypeptide encoded by
a polynucleotide that selectively hybridizes to a sequence that is
at least 90% identical to a sequence comprising at least 20
contiguous nucleotides of SEQ ID NO:1.
23. The kit of claim 20, wherein the reagents specific for
detection of anti-HE4 antibodies comprise a polypeptide comprising
a sequence at least 95% identical to a sequence comprising at least
10 contiguous amino acids of SEQ ID NO:2.
24. The kit of claim 20, wherein the polypeptide is attached to a
solid support.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date of
U.S. Provisional Application No. 61/377,387, which was filed on
Aug. 26, 2010. For the purpose of any U.S. application that may
claim the benefit of U.S. Provisional Application No. 61/377,387,
the contents of that earlier filed application are hereby
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions and methods
for assessing the risk, diagnosis and/or prognosis of subjects at
risk from or suffering from a cancer associated with human
epididymal four-disulfide core protein ("HE4") expressing tumors
and, in particular, to methods of measuring anti-HE4 antibodies for
use as an indicator of the presence of HE4 expressing cells, such
as ovarian tumor cells, and/or to determine the clinical status of
a patient undergoing treatment for a cancer associated with one or
more HE4 expressing tumors.
BACKGROUND
[0003] Ovarian carcinoma (OvC) is the second most frequent and the
most lethal gynecologic malignancy in the western world. Most cases
are diagnosed at an advanced stage, and this is reflected by a poor
prognosis with the overall five-year survival rate not exceeding
35%. Ovarian carcinoma is disproportionately deadly because
symptoms are vague and non-specific. Ovarian cancers shed malignant
cells into the naturally occurring fluid within the abdominal
cavity. These cells then have the potential to float in this fluid
and frequently implant on other abdominal (peritoneal) structures
including the uterus, urinary bladder, bowel, and lining of the
bowel wall (omentum). These cells can begin forming new tumor
growths before cancer is even suspected. More than 60% of patients
presenting with this disease already have stage III or stage IV
disease, when it has already spread beyond the ovaries, and more
than 75% of these patients die from disease, in spite of recent
improvements of chemotherapy for ovarian cancer. However, if
diagnosis is made early in the disease, five-year survival rates
can reach 90% to 98%.
[0004] One marker for ovarian cancer that is used in serum assays
for ovarian cancer is CA125 (Bast, R. C., et al., Gynecol. Oncol.
22:115-120 (1985); Einhorn, N., et al., Obstet. Gynaecol.
67:414-416 (1986); Einhorn, et al., Obstet. Gynecol. 80:14-18
(1992); Jacobs, I. J., et al., Br. Med. J. 313:1355-1358 (1996)).
However, while CA125 is found elevated in the majority of all
ovarian cancers, it is found in only half of those with early stage
disease (Hellstrom, I., et al., Cancer Research 63:3695-3700
(2003)). Moreover, CA125 is also elevated in several non-malignant
conditions (Fung, M. F., et al., J. Obstet. Gynaecol. Can.,
26:717-728 (2004); Mas, M. R., et al, Dig. Liver Dis. 32:595-597
(2000); Malkasian, G. D., et al, Am. J. Obstet. Gynecol.
159:341-346 (1988)), which can lead to a false positive result.
[0005] Thus, there is a great need to develop more effective tools
for detecting potentially curable, early stage malignant
conditions, such as ovarian carcinoma.
SUMMARY
[0006] In accordance with the foregoing, in one aspect, a method is
provided for detecting the presence of HE4-expressing cells in a
human subject comprising determining the presence or amount of
anti-HE4 antibodies in a biological sample obtained from the human
subject, wherein the presence or amount of anti-HE4 antibodies in
the biological sample is indicative of the presence of
HE4-expressing cells in the human subject. In some embodiments, the
presence of HE4-expressing cells in a human subject is indicative
that the subject is suffering from ovarian cancer, or is at risk
for developing a ovarian tumor.
[0007] In another aspect, a method is provided for monitoring the
efficacy of treatment of a human cancer patient undergoing
therapeutic treatment for an HE4-expressing tumor. The method
comprises: (a) providing a biological sample from a human patient
undergoing therapeutic treatment for a cancer associated with
HE4-expressing tumor cells; (b) determining the presence or amount
of anti-HE4 antibodies in the biological sample by contacting the
biological sample with a polypeptide encoded by a polynucleotide
that selectively hybridizes to a sequence at least 90% identical to
a sequence comprising at least 20 contiguous nucleotides of SEQ ID
NO:1; and (c) comparing the determined presence or amount of
anti-HE4 antibodies to an antibody reference standard, wherein an
amount of anti-HE4 antibody greater than the reference standard is
indicative of a positive response to the therapeutic treatment for
the cancer.
[0008] In another aspect, a kit is provided for detecting the
presence of HE4-expressing cells in a human subject, the kit
comprising reagents specific for detection of the presence or
amount of anti-HE4 antibodies in a biological sample obtained from
a human subject and printed instructions for comparison of the
detected presence or amount of anti-HE4 antibodies with a reference
standard.
DESCRIPTION OF THE DRAWINGS
[0009] The foregoing aspects and many of the attendant advantages
of this invention will become more readily appreciated as the same
become better understood by reference to the following detailed
description, when taken in conjunction with the accompanying
drawings, wherein:
[0010] FIG. 1 graphically illustrates the results of an ELISA assay
testing for the presence/amount of anti-HE4 antibodies from human
serum samples obtained from 10 patients with ovarian cancer, and 1
healthy control subject; as described in Example 2.
[0011] FIG. 2 depicts HE4 fragments expressed as fusion proteins
with coat protein pVIII using the primers shown in Table 1.
DETAILED DESCRIPTION
[0012] Unless specifically defined herein, all terms used herein
have the same meaning as they would to one skilled in the art of
the present invention.
[0013] The terms "percent identity" or "percent identical," as
applied to polypeptide sequences, such as the HE4 polypeptide or a
portion thereof, is defined as the percentage of amino acid
residues in a candidate protein sequence that are identical with
the subject protein sequence (such as the amino acid sequence set
forth in SEQ ID NO:2, or a portion thereof comprising at least 10
consecutive amino acid residues) after aligning the candidate and
subject sequences to achieve the maximum percent identity. For
example, percentage identity between two protein sequences can be
determined by pairwise comparison of the two sequences using the
bl2seq interface at the Web site of the National Center for
Biotechnology Information (NCBI), U.S. National Library of
Medicine, 8600 Rockville Pike, Bethesda, Md. 20894, U.S.A. The
bl2seq interface permits sequence alignment using the BLAST tool
described by Tatiana, A., et al, "Blast 2 Sequences--A New Tool for
Comparing Protein and Nucleotide Sequences," FEMS Microbiol. Lett.
174:247-250 (1999). The following alignment parameters are used:
Matrix=BLOSUM62; Gap open penalty=11; Gap extension penalty=1; Gap
x_dropff=50; Expect=10.0; Word size=3; and Filter=off.
[0014] The terms "percent identity" or "percent identical," as
applied to nucleic acid molecules, is the percentage of nucleotides
in a candidate nucleic acid sequence that are identical with a
subject nucleic acid molecule sequence (such as the nucleic acid
molecule sequence set forth in SEQ ID NO:1, or a portion thereof
comprising at least 20 consecutive nucleotides) after aligning the
sequences to achieve the maximum percent identity, and not
considering any nucleic acid residue substitutions as part of the
sequence identity. No gaps are introduced into the candidate
nucleic acid sequence in order to achieve the best alignment.
Nucleic acid sequence identity can be determined in the following
manner. The subject polynucleotide molecule sequence is used to
search a nucleic acid sequence database, such as the Genbank
database, using the program BLASTN version 2.1 (based on Altschul,
et al., Nucleic Acids Research 25:3389-3402 (1997)). The program is
used in the ungapped mode. Default filtering is used to remove
sequence homologies due to regions of low complexity as defined in
Wootton, J. C., and S. Federhen, Methods in Enzymology 266:554-571
(1996). The default parameters of BLASTN are utilized.
[0015] As used herein, the term "healthy human subject" refers to
an individual who is known not to suffer from cancer, such
knowledge being derived from clinical data on the individual
including, but not limited to, a different cancer assay to that
described herein. The healthy individual is also preferably
asymptomatic with respect to the early symptoms associated with
HE4-expressing tumors such as ovarian cancer, which include, for
example, rectal pressure, abdominal bloating, and swelling; and is
also preferably asymptomatic with respect to other reproductive
diseases or conditions.
[0016] As used herein, the term "HE4-expressing tumor" refers to
any type of cancer cells and/or tumors that are identified as
having a neoplastic condition associated with an increased
expression of HE4, wherein HE4 refers to at least one of SEQ ID
NO:1, SEQ ID NO:2 and mammalian homologs thereof, or a fragment
thereof comprising at least ten consecutive residues of the protein
(SEQ ID NO:2), or at least 20 consecutive nucleotides of the cDNA
(SEQ ID NO:1), as compared to normal tissues, including but not
limited to, ovarian cancer, lung adenocarcinoma (Bingle et al.,
Respiratory Research 7:61-80 (2006), and salivary gland tumors.
[0017] As used herein, the term "ovarian cancer" refers to any type
of ovarian cancer including, but not limited to, serous ovarian
cancer, non-invasive ovarian cancer, mixed phenotype ovarian
cancer, mucinous ovarian cancer, endometrioid ovarian cancer, clear
cell ovarian cancer, papillary serous ovarian cancer, Brenner cell,
and undifferentiated adenocarcinoma.
[0018] As used herein, the term "recurrence of a tumor expressing
HE4" refers to clinical evidence of cancer related to cells
expressing HE4, for example, ovarian cancer, or tumor cells derived
therefrom based upon clinical data on the individual including, but
not limited to, a different cancer assay to that described
herein.
[0019] As used herein, the term "good prognosis" in the context of
cancer associated with one or more HE4-expressing tumors (e.g.,
ovarian cancer) refers to patients who are likely to be cured from
their disease, or to have at least a five-year tumor-free survival
period following the initial diagnosis.
[0020] As used herein, the term "poor prognosis" in the context of
cancer associated with one or more HE4-expressing tumors (e.g.,
ovarian cancer) refers to patients who are likely to die from their
disease within a five-year period following the initial
diagnosis.
[0021] The human epididymal four-disulfide core protein ("HE4")
(SEQ ID NO:2) is encoded by the mRNA sequence set forth as SEQ ID
NO:1 (which corresponds to Genbank Accession No. AY212888). HE4
cDNA was first isolated from human epididymis (Kirchhoff et al.,
1991), and HE4 cDNA was later detected with high frequency in cDNA
libraries constructed from ovarian carcinomas (Wang et al., Gene
229:101 (1999)). The HE4 protein belongs to the "four-disulfide
core" family of proteins, which comprises a heterogeneous group of
small acid and heat stable molecules of divergent function,
referred to as "soluble HE4-related peptides" (SHRP) (Kirchhoff, et
al., Biol. Reprod. 45:350-357 (1991). The conserved spacing of
eight core cysteine residues in the amino acid sequences of the
four-disulfide core family member polypeptides (aa regions 32-73
and 76-123 of SEQ ID NO:2) is thought to direct the folding of
these molecules into a compact and stable structure. Many members
of the four-disulfide core family are protease inhibitors; however,
for some family members, including HE4, no function has yet been
identified.
[0022] As used herein, the term "HE4" protein encompasses naturally
occurring HE4 protein that is isolated from a biological sample
obtained from a human subject as well as HE4 protein isolated from
cultured cells making HE4 (e.g., cultured ovarian carcinoma cells),
or made by recombinant DNA technology (e.g., in eukaryotic
expression systems (e.g., COS cells)), in yeast, mammalian, or in
bacterial expression systems.
[0023] In accordance with the foregoing, the present inventors have
generated a reproducible assay for detecting antibodies to native
HE4 protein (SEQ ID NO:2) in a biological sample (e.g., plasma or
serum), as described in Example 2. As further described in Examples
2-3, the inventors have used this assay to detect the presence of
antibodies to HE4 in human subjects, and have found that subjects
clinically identified as suffering from ovarian cancer had a
significantly higher incidence of anti-HE4 antibodies as compared
to normal healthy female subjects.
[0024] In accordance with the foregoing, in one aspect, a method is
provided for detecting the presence of HE4-expressing cells in a
human subject. The method comprises determining the presence or
amount of anti-HE4 antibodies in a biological sample obtained from
the human subject, wherein the presence or amount of anti-HE4
antibodies in the biological sample is indicative of the presence
of HE4-expressing cells in the human subject. In one embodiment,
the presence or amount of anti-HE4 antibodies in the biological
sample is determined by contacting the biological sample with a
polypeptide encoded by a polynucleotide that selectively hybridizes
to a sequence at least 80% identical, or at least 90% identical, or
at least 95% identical, or at least 98% identical, or at least 99%
identical to a sequence comprising at least 20 contiguous
nucleotides of SEQ ID NO:1. In some embodiments, the presence or
amount of anti-HE4 antibody in the biological sample is determined
by contacting the biological sample with a polypeptide comprising a
sequence at least 80% identical, or at least 90% identical, or at
least 95% identical, or at least 98% identical, or at least 99%
identical to a sequence comprising at least 10 contiguous amino
acids of SEQ ID NO:2. In one embodiment, the presence or amount of
anti-HE4 antibodies in comparison to a reference standard (e.g., a
negative control) is indicative of the presence of HE4-expressing
cells, such as tumor cells, in the human subject. In another
embodiment, the amount of anti-HE4 antibodies over a predetermined
threshold amount is indicative of the presence of HE4-expressing
cells in a human subject. In some embodiments of the method, the
presence of HE4-expressing cells in a human subject is indicative
that the subject is suffering from or at risk for developing
ovarian cancer.
[0025] A wide variety of biological samples may be used in the
methods of the invention, including biological fluids. Non-limiting
examples of biological fluids include blood, plasma, serum, ascitic
fluid, urine, saliva, tears, pleural fluid, sputum, vaginal fluid
(discharge), and washings obtained during a medical procedure
(e.g., pelvic or other washings obtained during biopsy, endoscopy,
or surgery).
[0026] The methods of this aspect of the invention may be used as a
diagnostic tool to distinguish between a subject suffering from a
disease or condition associated with the expression of HE4 and a
disease or condition not associated with the expression of HE4. In
one embodiment of the method, a biological sample is obtained from
a human subject suffering from at least one symptom associated with
ovarian cancer. Symptoms associated with ovarian cancer are known
to those of skill in the field of medicine. Non-limiting examples
of such symptoms include abdominal swelling/bloating,
abdominal/pelvic pain or pressure, gastrointestinal symptoms (e.g.,
gas, indigestion, nausea, or changes in bowel movements), vaginal
bleeding or discharge, urinary problems (e.g., urgency, burning or
spasms), fatigue, fever, back pain, and difficulty breathing.
[0027] In some embodiments, the methods of this aspect of the
invention further comprise performing at least one additional
diagnostic assay to determine if the subject with anti-HE4
antibodies has an ovarian tumor. In some embodiments, the methods
of this aspect of the invention further comprise performing at
least one additional diagnostic assay for ovarian cancer on the
subject, such as, for example, detecting the presence of CA125 in a
biological sample, ultrasound, CT scan, MRI scan, biopsy, aspirate,
and the like.
[0028] As described in more detail herein, in some embodiments, the
methods of the invention that include the detection of antibodies
to native HE4 may be used and optionally combined with an assay to
detect SHRP antigen, in order to detect the presence of
HE4-expressing tumor cells, to determine the presence or likelihood
of recurrence of a cancer associated with an HE4-expressing tumor,
such as ovarian cancer, to assess the clinical status and/or
prognosis of a patient suffering from a cancer associated with
HE4-expressing tumors, and/or to monitor the efficacy of treatment
of cancer in a patient. The amount of SHRP antigen detected in the
biological sample may be compared to a reference standard such as
an antigen reference value, wherein detection of an increased
amount of SHRP antigen in the sample as compared to the reference
standard is indicative of the presence of HE4-expressing tumor
cells in the human subject.
[0029] In another embodiment, a method is provided for monitoring
the efficacy of treatment of a human cancer patient undergoing
therapeutic treatment for an HE4-expressing tumor. The method
comprises: (a) providing a biological sample from a human patient
undergoing therapeutic treatment for a cancer associated with an
HE4-expressing tumor; (b) determining the presence or amount of
anti-HE4 antibodies in the biological sample by contacting the
biological sample with a polypeptide encoded by a polynucleotide
that selectively hybridizes to a sequence at least 90% identical to
a sequence comprising at least 20 contiguous nucleotides of SEQ ID
NO:1; and (c) comparing the determined presence or amount of
anti-HE4 antibodies to an antibody reference value wherein an
amount of anti-HE4 antibody greater than the antibody reference
value is indicative of a positive response to the therapeutic
treatment for the cancer.
[0030] In another embodiment, a method is provided for determining
the likelihood of recurrence of an HE4-expressing tumor in a human
patient undergoing therapeutic treatment for a cancer associated
with an HE4-expressing tumor. The method comprises: (a) providing a
biological sample from a human patient undergoing therapeutic
treatment for a cancer associated with an HE4-expressing tumor; (b)
determining the presence or amount of anti-HE4 antibodies in the
biological sample by contacting the biological sample with a
polypeptide encoded by a polynucleotide that selectively hybridizes
to a sequence at least 80%, such as at least 90% identical to a
sequence comprising at least 20 contiguous nucleotides of SEQ ID
NO:1; and (c) comparing the presence or amount of anti-HE4
antibodies determined in step (b) to an antibody reference value,
wherein an amount of anti-HE4 antibody greater than the antibody
reference value is indicative of a lower risk of HE4-expressing
tumor recurrence and wherein an amount of anti-HE4 antibody lower
than the reference value is indicative of greater risk of
HE4-expressing tumor recurrence in the human patient.
[0031] In accordance with one embodiment of the methods of the
invention, a human patient undergoing therapeutic treatment for a
cancer associated with an HE4-expressing tumor is assessed for
their clinical status and likelihood of recurrence of cancer. The
methods in accordance with this embodiment may be practiced with
patients previously diagnosed and treated for an HE4-expressing
tumor, such as ovarian cancer, lung adenocarcinoma, or a salivary
gland tumor (e.g., treated with surgery and/or previously or
currently undergoing therapeutic treatment, such as chemotherapy,
radiation therapy, protein therapeutics, including antibodies, gene
therapy, cancer vaccine therapy, stem cell transplant, or other
therapy). Recurrence of ovarian cancer is a clinical recurrence as
determined by the presence of one or more clinical symptoms of an
ovarian cancer, such as, for example, a metastases, or
alternatively, as determined in a biochemical test, immunological
test, or serological test such as, for example, a cross-reactivity
in a biological sample to a CA125 antibody, or other diagnostic
test. Preferably, the recurrence of ovarian cancer is capable of
being detected at least about 2 years from treatment, more
preferably about 2-3 years from treatment, and even more
preferably, about 4 or 5 or 10 years from treatment.
[0032] A 1-4 staging system is used for describing ovarian cancer,
as set forth by the International Federation of Gynecology and
Obstetrics ("FIGO") staging system, which uses information obtained
after surgery. Surgeries can include a total abdominal
hysterectomy, removal of one or both ovaries and fallopian tubes,
the omentum, and/or pelvic washings for cytology.
[0033] Stage I--limited to one or both ovaries [0034] IA--involves
one ovary; capsule intact; no tumor on ovarian surface; no
malignant cells in ascites or peritoneal washings [0035]
IB--involves both ovaries; capsule intact; no tumor on ovarian
surface; negative washings [0036] IC--tumor limited to ovaries with
any of the following: capsule ruptured, tumor on ovarian surface,
positive washings
[0037] Stage II--pelvic extension or implants [0038] IIA--extension
or implants onto uterus or fallopian tube; negative washings [0039]
IIB--extension or implants onto other pelvic structures; negative
washings [0040] IIC--pelvic extension or implants with positive
peritoneal washings
[0041] Stage III--microscopic peritoneal implants outside of the
pelvis; or limited to the pelvis with extension to the small bowel
or omentum [0042] IIIA--microscopic peritoneal metastases beyond
pelvis [0043] IIIB--macroscopic peritoneal metastases beyond pelvis
less than 2 cm in size [0044] IIIC--peritoneal metastases beyond
pelvis >2 cm or lymph node metastases, note: para-aortic lymph
node metastases are considered regional lymph nodes
[0045] Stage IV--distant metastases--in the liver, or outside the
peritoneal cavity
[0046] In accordance with some embodiments of the invention, a
biological sample is obtained from a human patient (previously
diagnosed with and previously treated for ovarian cancer, or
currently undergoing treatment for ovarian cancer) and is assayed
for the presence or concentration of anti-HE4 antibodies.
Biological samples for use in the methods of the invention include
biological fluids. Non-limiting examples of biological fluids
include blood, plasma, serum, ascitic fluid, urine, saliva, tears,
pleural fluid, sputum, vaginal fluid (discharge), and washings
obtained during a medical procedure (e.g., pelvic or other washings
obtained during biopsy, endoscopy, or surgery). The ability to use
a sample of biological fluid to assess the clinical status of a
subject with regard to an HE4-expressing tumor (such as ovarian
cancer or other HE4-expressing tumors), provides relative ease as
compared to obtaining a tissue biopsy sample of a tumor. Moreover,
it enables monitoring of a patient during and/or post-treatment
and, importantly, allows for earlier detection of recurrence and/or
progression of ovarian cancer (or other HE4-expressing tumors).
[0047] In accordance with the methods of this aspect of the
invention, the concentration of anti-HE4 antibody is measured in a
biological sample obtained from a human patient. Any immunoassay
may be used to measure the concentration of anti-HE4 antibody; for
example, enzyme-linked immunosorbent assays (ELISA) and
radioimmunoassays (RIA), western blotting, FACS analysis, and the
like. More preferably, the assay will be capable of generating
quantitative results. The biological sample may be diluted in a
suitable buffer prior to analysis, for example, the sample may be
diluted by a factor of at least 1:2, 1:5, 1:10, 1:20, 1:30, 1:40,
1:50, 1:80, 1:100, 1:200 or greater.
[0048] In one embodiment, the presence or amount of anti-HE4
antibody in the biological sample is determined by contacting the
biological sample with an HE4 polypeptide encoded by a
polynucleotide that selectively hybridizes to a sequence at least
80% identical (e.g., at least 85% identical, or at least 90%
identical, or at least 95% identical, or at least 99% identical) to
SEQ ID NO:1, or a fragment thereof comprising at least 20
consecutive nucleotides (or at least 25 or 30, or at least 40, 60,
or 80 consecutive nucleotides) of SEQ ID NO:1.
[0049] In another embodiment, the presence or amount of anti-HE4
antibody in the biological sample is determined by contacting the
biological sample with an HE4 polypeptide at least 80% identical
(e.g., at least 85% identical, or at least 90% identical, or at
least 95% identical, or at least 99% identical) to the human
soluble HE4-related protein provided as SEQ ID NO:2, or a fragment
thereof comprising at least 10 consecutive amino acid residues, (or
at least 20 or at least 30, such as at least 50 consecutive amino
acid residues) of SEQ ID NO:2.
[0050] A fragment of an HE4 polypeptide has a amino acid sequence
contains fewer amino acids than the full-length HE4 amino acid
sequence as set forth in SEQ ID NO: 2. In some embodiments, the
fragment can include the N-terminal domain, N-WFDC. As shown in
FIG. 2, N-WFDC (SEQ ID NO: 5) extends from amino acid 31 to amino
acid 75 of SEQ ID NO: 2. In some embodiments, the fragment can
include the C-terminal domain C-WFDC. As shown in FIG. 2, C-WFDC
(SEQ ID NO: 6) extends from amino acid 76 to amino acid 124 of SEQ
ID NO: 2. Other exemplary fragments of HE4 can include polypeptides
having an amino acid sequence corresponding to the amino acid
sequence extending from positions 31-52 (SEQ ID NO: 7); positions
42-63 (SEQ ID NO: 8); positions 53-75 (SEQ ID NO: 9); positions
76-100 (SEQ ID NO: 10); positions 89-112 (SEQ ID NO: 11); positions
89-112 (SEQ ID NO: 12); or positions 101-124 (SEQ ID NO: 13) of SEQ
ID NO: 2. In some embodiments, fragments of HE4 can span the N-WFDC
and the C-WFDC domains. Exemplary fragments include polypeptides
having an amino acid sequence corresponding to the amino acid
sequence extending from positions 53-100 of SEQ ID NO: 2. A
fragment comprising at least 20 consecutive nucleotides (or at
least 25 or 30, or at least 40, 60, or 80 consecutive nucleotides)
of SEQ ID NO:1 can be, for example, a fragment of SEQ ID NO: 1 that
encodes SEQ ID NO: 5-13.
[0051] The anti-HE4 antibodies or the invention specifically bind
to an epitope on the HE4 polypeptide. An epitope refers to an
antigenic determinant on a target that is specifically bound by the
paratope, i.e., the binding site of an antibody. Epitopic
determinants usually consist of chemically active surface groupings
of molecules such as amino acids or sugar side chains, and
typically have specific three-dimensional structural
characteristics, as well as specific charge characteristics.
Epitopes generally have between about 4 to about 10 contiguous
amino acids (a continuous epitope), or alternatively can be a set
of noncontiguous amino acids that define a particular structure
(e.g., a conformational epitope). Thus, an epitope can consist of
at least 4, at least 6, at least 8, at least 10, and at least 12
such amino acids. Methods of determining the spatial conformation
of amino acids are known in the art, and include, for example,
x-ray crystallography and 2-dimensional nuclear magnetic
resonance.
[0052] The binding of the anti-HE4 antibodies can be analyzed using
standard methods. Method of mapping epitopes was well known in the
art. The type of epitopes, i.e. linear or conformational dependent
epitopes, can be determined by comparing the reactivity of the
antibodies towards denatured and reduced HE4 with that of the
antibodies against HE4 that has not been denatured and reduced.
Reactivity of the anti-HE4 antibodies against phage fusion proteins
can be used to identify binding to specific fragments of HE4. Such
methods are described in Example 4 and in PCT/US11/25321, which is
herein incorporated by reference in its entirety.
[0053] Specifically binding antibodies are can be antibodies that
1) exhibit a threshold level of binding activity; and/or 2) do not
significantly cross-react with known related polypeptide molecules.
The binding affinity of an antibody can be readily determined by
one of ordinary skill in the art, for example, by Scatchard
analysis (Scatchard, Ann. NY Acad, Sci. 51:660-672, 1949).
[0054] In some embodiments the anti-HE4 antibodies can bind to
their target epitopes or mimetic decoys at least 1.5-fold, 2-fold,
5-fold 10-fold, 100-fold, 10.sup.3-fold, 10.sup.4-fold,
10.sup.5-fold, 10.sup.6-fold or greater for HE4 than to other
proteins predicted to have some homology to HE4.
[0055] In some embodiments the anti-HE4 antibodies bind with high
affinity of 10.sup.-4M or less, 10.sup.-7M or less, 10.sup.-9M or
less or with subnanomolar affinity (0.9, 0.8, 0.7, 0.6, 0.5, 0.4,
0.3, 0.2, 0.1 nM or even less). In some embodiments the binding
affinity of the antibodies is at least 1.times.10.sup.6 Ka, at
least 5.times.10.sup.6 Ka, at least 1.times.10.sup.7 Ka, at least
2.times.10.sup.7 Ka, at least 1.times.10.sup.8 Ka, or greater.
Antibodies may also be described or specified in terms of their
binding affinity to HE4. In some embodiments binding affinities
include those with a Kd less than 5.times.10.sup.-2 M, 10.sup.-2 M,
5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.-3M, 10.sup.-4 M,
5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M,
5.times.10.sup.-7 M, 10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8M,
5.times.10.sup.-9M, 5.times.10.sup.-10 M, 10.sup.-10 M,
5.times.10.sup.-11 M, 10.sup.-11M, 5.times.10.sup.-12M, 10.sup.-12
M, 5.times.10.sup.-13 M, 10.sup.-13 M, 5.times.10.sup.-14M,
10.sup.-14M, 5.times.10.sup.-15M, or 10.sup.-15M, or less.
[0056] In one embodiment, the anti-HE4 antibody presence or amount
is measured in the biological sample through the use of an ELISA
assay. Standard solid phase ELISA formats are particularly useful
in determining the concentration of a protein or antibody from a
variety of biological samples, such as serum. In one form, such an
assay involves immobilizing an HE4 polypeptide or fragment thereof
onto a solid matrix, such as, for example, a polystyrene or
polycarbonate microwell or dipstick, a membrane, or a glass support
(e.g., a glass slide). For example, an HE4-coated well of an ELISA
plate may be utilized. The biological sample is contacted with the
HE4-coated well, and the anti-HE4 antibody in the sample is bound
and captured. After binding and washing to remove non-specifically
bound immune complexes, the antibody-antigen complex is detected.
Detection may be carried out with any suitable method, such as the
addition of a second antibody linked to a label.
[0057] In accordance with various embodiments of the methods of
this aspect of the invention, an anti-HE4 antibody reference value
may be obtained from a control group of apparently healthy
subjects, for example, as described in EXAMPLES 2 and 3. In some
embodiments, the antibody reference value is determined in an ELISA
assay using serum obtained from healthy subjects diluted at least
1:20. In one embodiment, the antibody reference value is determined
using serum obtained from patients diagnosed with and/or previously
treated for a cancer comprising HE4-expressing tumor cells. An
exemplary ELISA assay for detecting anti-HE4 antibody levels in
blood samples is described in EXAMPLE 2.
[0058] In accordance with the prognostic applications of the
invention, in one embodiment the level of anti-HE4 antibody in a
biological sample obtained from an ovarian cancer patient is then
compared to the antibody reference value. If the antibody
concentration in the patient tested is higher than the reference
value, such as at least 1.5-fold, more preferably at least two-fold
or higher, with a P value of less than 0.05, and the patient has
previously undergone treatment for ovarian cancer, then the patient
has a reduced likelihood of recurrence of ovarian cancer. If the
antibody concentration in an ovarian cancer patient is lower than
the reference value, such as at least 1.5-fold or two-fold or
lower, with a P value of less than 0.05, and the patient has
previously undergone treatment for ovarian cancer, then the patient
has an increased likelihood of recurrence of ovarian cancer. In
another embodiment, the presence of anti-HE4 antibody is determined
by comparison to a negative antibody control sample and optionally
also to a positive antibody control sample.
[0059] In another aspect, the invention provides a method of
assessing the prognosis of a human cancer patient suffering from an
HE4-expressing tumor. The method comprises: (a) determining the
presence or amount of anti-HE4 antibodies in a biological sample
from a human patient suffering from an HE4-expressing tumor by
contacting the biological sample with a polypeptide encoded by a
polynucleotide that selectively hybridizes to a sequence that is at
least 80% identical, such as at least 90% identical to a sequence
comprising at least 20 contiguous nucleotides of SEQ ID NO:1; (b)
determining the presence or amount of soluble HE4-related peptides
(SHRP) encoded by a polynucleotide that selectively hybridizes to a
sequence at least 80%, such as at least 90% identical to a sequence
comprising at least 20 contiguous nucleotides of SEQ ID NO:1 in a
biological sample from the human patient tested in step (a); and
(c) comparing the amount of anti-HE4 antibodies determined in step
(a) to an antibody reference level, and comparing the amount of
SHRP determined in step (b) to an antigen reference level, wherein
the detection of SHRP in the sample at a lower amount than the
antigen reference level, in combination with the detection of
anti-HE4 antibodies in the sample at a higher amount than the
antibody reference level, is indicative of a good prognosis for the
patient.
[0060] In accordance with this aspect of the invention, the method
comprises the step of determining the presence and/or amount of
SHRP in a biological sample obtained from a patient suffering from
an HE4-expressing tumor, such as an ovarian cancer patient. As
described above, SHRP is a soluble protein that has been found in
the circulation of both healthy and cancer patients. The presence
or amount of SHRP may be determined using any assay capable of
detecting and/or measuring the amount of SHRP polypeptide.
[0061] In one embodiment, the concentration of an SHRP polypeptide
encoded by a polynucleotide that selectively hybridizes to a
sequence at least 80% identical (e.g., at least 85% identical, or
at least 90% identical, or at least 95% identical, or at least 99%
identical) to SEQ ID NO:1, or a fragment thereof comprising at
least 20 consecutive nucleotides (or at least 25 or 30, or at least
40, 60, or 80 consecutive nucleotides) of SEQ ID NO:1, is measured
in the biological sample.
[0062] In another embodiment, the amount of an SHRP polypeptide at
least 80% identical (e.g., at least 85% identical, or at least 90%
identical, or at least 95% identical, or at least 99% identical) to
the human soluble HE4-related protein provided as SEQ ID NO:2, or a
fragment thereof comprising at least 10 consecutive amino acid
residues (or at least 20 or at least 30, such as at least 50
consecutive amino acid residues) of SEQ ID NO:2 is measured in the
biological sample.
[0063] The concentration and/or relative amount, or detection of
soluble HE4-related protein (SHRP) present in a biological fluid
sample may be determined using any convenient method for measuring
SHRP including, but not limited to, ELISA, radioimmunoassay,
chemiluminescence assay, immunofluorescence staining and the like
that include an antibody that specifically binds to SHRP. Other
protein detection methods may also be used to measure SHRP,
including mass spectroscopy, western blot, FACS, and the like.
Suitable biological samples include a biological fluid selected
from the group consisting of blood, plasma, serum, ascitic fluid,
and urine.
[0064] Specific antibodies, including monoclonal antibodies
directed against SHRP and variants thereof, can be readily prepared
using conventional techniques, and may be used in such methods. For
example, a double determinant ("sandwich") ELISA assay using two
MAbs 2H5 and 3D8 (which recognize two different epitopes on the
same antigen) may be used to detect SHRP in sera, as described in
Hellstrom, I., et al., Cancer Research 63:3695-3700 (2003). Other
ELISA assays may be used to detect one or more variants of HE4
using antibodies described above, or other antibodies against
HE4.
[0065] In accordance with various embodiments of the methods of
this aspect of the invention, an SHRP antigen reference value may
be obtained from a control group of apparently healthy subjects;
for example, as described in Example 3. In some embodiments, the
antigen reference value is determined in an ELISA assay using serum
obtained from healthy subjects. For example, in a serum sample, the
serum may be diluted up to 1:100 and measured in an ELISA assay,
where a negative control obtained from a healthy subject gives an
absorbance value of <0.2 and a positive control obtained from a
ovarian cancer patient gives an absorbance value of >0.2. See
Hellstrom, I., et al., Cancer Research 63:3695-3700 (2003).
Absorbance values may be determined by any method known in the art.
For example, absorbance of light at 450 nanometers, often referred
to as the optical density (OD), is commonly used. In one
embodiment, the antigen reference value is determined using serum
obtained from patients diagnosed with and/or previously treated for
a cancer comprising HE4-expressing tumor cells.
[0066] In some embodiments, the methods of the invention further
comprise the step of determining levels of another ovarian cancer
marker, such as integrin-linked kinase (INK), CA125, TADG-12,
kallikrein 10, prostasin, osteopontin, creatine kinase beta,
serotransferrin, neutrophil-gelatinase associated lipocalin (NGAL),
CD163, or Gc-globulin in a biological sample obtained from the
subject. The second marker may be detected at the DNA, RNA, or
protein level using conventional methods known in the art.
[0067] In another aspect, the invention provides a method of
monitoring the efficacy of treatment of a human patient diagnosed
with an HE4-expressing tumor. The method comprises: (a) determining
a first concentration of HE4-mesothelin antibodies in a first
biological sample taken from a human patient diagnosed with an
HE4-expressing tumor prior to initiation of treatment for cancer;
(b) determining a second concentration of anti-HE4 antibodies in a
second biological sample from the human patient taken after
initiation of treatment for cancer; and (c) comparing the first and
second concentrations of anti-HE4 antibodies, wherein an increase
in the second concentration of anti-HE4 antibodies as compared to
the first concentration of anti-HE4 antibodies measured in the
first biological sample indicates a positive response to the
treatment for cancer.
[0068] In accordance with the method of this aspect of the
invention, a first biological sample is taken from a cancer patient
before initiation of treatment, and a second biological sample is
taken from the patient at least one time after initiation of
treatment. In some embodiments, plural treated biological samples
from the subject (e.g., a subject in a preclinical trial) are taken
over periodic intervals of time after initiation of treatment.
[0069] As used herein, the term "treatment" refers to surgical
intervention or to the administration of one or more cancer
inhibitory agents for the alleviation of symptoms associated with
cancer, or halt of further progression or worsening of the
symptoms. For example, successful treatment may include a removal
of a tumor, such as an HE4-expressing tumor; an alleviation of
symptoms or halting the progression of the disease, as measured by
a reduction in the growth rate of a tumor; a halt in the growth of
a tumor; a reduction in size of the tumor; partial or complete
remission of the cancer; or increased survival or clinical benefit.
For example, treatment of a subject suffering from an
HE4-expressing tumor may include one or more of the following:
surgery to remove one or more tumors and/or administration of a
therapeutic agent, such as chemotherapy, radiation therapy, protein
therapeutics (e.g., antibodies, gene therapy, cancer vaccine
therapy, stem cell transplant, or other therapy).
[0070] For example, with regard to treatment for ovarian cancer,
surgery is a preferred treatment. The type of surgery depends upon
how widespread the cancer is when diagnosed (the cancer stage), as
well as the type and grade of cancer. The surgeon may remove one
(unilateral oophorectomy) or both ovaries (bilateral oophorectomy),
the fallopian tubes (salpingectomy), and the uterus (hysterectomy).
For some very early tumors (stage 1, low grade or low-risk
disease), only the involved ovary and fallopian tube will be
removed (called a "unilateral salpingo-oophorectomy," or "USO"),
especially in young females who wish to preserve their fertility.
In advanced stages of disease, as much tumor as possible is removed
(debulking surgery). In cases where this type of surgery is
successful, the prognosis is improved compared to patients where
large tumor masses (more than 1 cm in diameter) are left behind.
Chemotherapy is typically used after surgery to treat any residual
disease. Chemotherapeutic agents, such as a platinum derivative
(e.g., taxane) may be administered systemically, or may be
administered intra-peritoneally via direct infusion into the
abdominal cavity. Other examples of therapeutic agents for use in
treatment of ovarian cancer include, but are not limited to,
protein therapeutics (e.g., antibodies), gene therapy, cancer
vaccine therapy, and stem cell transplants. The methods of this
aspect of the invention may also be used to measure the efficacy of
candidate therapeutic agents for treatment of ovarian cancer.
[0071] The methods of this aspect of the invention may also be used
to determine the clinical status of a patient after undergoing a
treatment, such as surgery to remove a tumor. In accordance with
this embodiment, the level of anti-HE4 antibody in a biological
sample obtained from a cancer patient that has been treated for an
HE4-expressing tumor is then compared to the antibody reference
value. If the antibody concentration in the patient tested is
higher than the reference value, such as at least 1.5-fold, more
preferably at least two-fold or higher, with a P value of less than
0.05, then the patient's clinical status is expected to be improved
with the treatment (i.e., the patient has a reduced likelihood of
recurrence of ovarian cancer). If the antibody concentration in the
treated cancer patient is lower than the reference value, such as
at least 1.5-fold or two-fold or lower, with a P value of less than
0.05, then the patient's clinical status is not expected to be
improved with the treatment (i.e., the patient has an increased
likelihood of recurrence of ovarian cancer).
[0072] In another aspect, a kit is provided for detecting the
presence of HE4-expressing cells in a human subject. The kit
comprises reagents specific for detection of anti-HE4 antibodies in
a biological sample obtained from a human subject and printed
instructions for comparison of the detected presence or amount of
anti-HE4 antibodies with a reference standard. The methods for
detection of anti-HE4 antibodies described herein may be performed
using the kits of the invention. In one embodiment, the kit
comprises a detection reagent for detecting anti-HE4 antibodies
comprising an HE4 polypeptide encoded by a polynucleotide that
selectively hybridizes to a sequence that is at least 80%
identical, or at least 90% identical, or at least 95% identical, or
at least 98% identical, or at least 99% identical to a sequence
comprising at least 20 contiguous nucleotides of SEQ ID NO:1. In
some embodiments, the kit comprises a detection reagent for
detecting anti-HE4 antibodies comprising an HE4 polypeptide that is
at least 80% identical, or at least 90% identical, or at least 95%
identical, or at least 98% identical, or at least 99% identical to
an amino acid sequence comprising at least 10 contiguous amino
acids of SEQ ID NO:2. In some embodiments, the kit comprises HE4
polypeptide, or fragment thereof, that is immobilized onto a solid
matrix, such as, for example, a polystyrene or polycarbonate
microwell or dipstick, a membrane, or a glass support (e.g., a
glass slide). For example, an HE4-coated well of an ELISA plate may
be utilized, wherein the biological sample is contacted with the
HE4-coated well and the anti-HE4 antibody in the sample is bound
and captured.
[0073] In some embodiments, the kit farther comprises a reference
standard selected from the group consisting of a specific numerical
threshold, a negative control sample for concurrent evaluation, or
statistical information correlating the amount of anti-HE4
antibodies detected with the likelihood of the presence of
HE4-expressing cancer cells in the subject. In some embodiments,
the reference standard is a negative control sample, and wherein
the negative control sample is included in the kit.
[0074] In preferred embodiments, the methods and kits of the
invention are capable of use at a point-of-care location, such as a
medical clinic (e.g., doctor's office) or hospital, in order to
rapidly obtain test results. Point-of-care testing (POCT) refers to
any hospital or medical clinic (doctor's office) employee
performing any type of laboratory test outside of the central
laboratory. POCT has revolutionized the continuum of patient care
process by providing laboratory results efficiently at the
patient's bedside for various tests such as HIV testing, urine
dipstick, etc. For example, rapid tests to detect HIV antibodies
have been developed that demonstrate sensitivities and
specificities comparable to those of enzyme immunoassays without
the need for sophisticated laboratory equipment and highly-trained
technicians. POCT can be used with unprocessed whole blood or oral
fluid specimens. See Branson, B. M., J. Lab. Medicine
27(7/8):288-295 (2003). POCT assays may be in any assay format that
allows for rapid testing, such as particle agglutination,
immunoconcentration and immunochromatography.
[0075] For example, particle agglutination POCT assays for
detecting anti-HE4 antibodies may be carried out by mixing a
patient specimen containing anti-HE4 antibodies with latex
particles coated with HE4 polypeptide (antigen), and if anti-HE4
antibody is present, cross-linking occurs within 10 to 60 minutes
and results in agglutination, with results interpreted
visually.
[0076] In another example of a POCT assay format for detecting
anti-HE4 antibodies, an immunoconcentration device (flow through)
may be used which employs solid-phase capture technology, which
involves the immobilization of HE4 polypeptides (antigen) on a
porous membrane. The patient specimen flows through the membrane
and is absorbed into an absorbent pad. If anti-HE4 antibodies are
present in the specimen, a dot or a line visibly forms on the
membrane when developed with a signal reagent (e.g., a colloidal
gold or selenium conjugate). A procedural control may also be
included on the membrane.
[0077] In yet another example of a POCT assay format to detect
anti-HE4 antibodies, immunochromatographic (lateral flow) strips
may be used that incorporate both antigen (HE4) and signal reagent
into a nitrocellulose strip. The patient specimen is applied to an
absorbent pad, or the specimen may be diluted in a vial of buffer
into which the test device is inserted. The specimen migrates
through the strip and combines with the signal reagent. A positive
reaction results in a visual line on the membrane where the HE4
antigen has been applied. A procedural control line may be applied
to the strip beyond the HE4 antigen line.
[0078] The following examples merely illustrate the best mode now
contemplated for practicing the invention, but should not be
construed to limit the invention.
Example 1
[0079] This Example describes the production and purification of
recombinant Human Epididymis Protein 4 (HE4) protein in Chinese
Hamster Ovary (CHO) cells.
[0080] Methods:
[0081] HE4-CIHDpa Plasmid Construction
[0082] Recombinant HE4 protein (SEQ ID NO:2) was generated as
follows.
[0083] A cDNA fragment encoding the Human Epididymis Protein 4
(HE4) (SEQ ID NO:2) was amplified by high fidelity polymerase
chain-reaction using the following primers:
[0084] The forward sense primer:
[0085] 5'-AAAAACCGGTATGCCTGCTTGTCGCCTAGG-3', (SEQ ID NO:3) was
designed to introduce an Age I restriction enzyme recognition site
at the 5' end of the gene appropriate for cloning.
[0086] The reverse antisense primer:
[0087] 5'-AAAACCTGCAGGTCAGAAATTGGGAGTGACAC-3', (SEQ ID NO: 4), was
designed to introduce an Sbf I restriction enzyme recognition site
at the 3' end of the gene appropriate for cloning.
[0088] The amplified DNA fragment containing the HE4 cDNA was
digested with Age I and Sbf I, and cloned into the CIHDpa mammalian
expression vector (by replacing the IFN-.gamma. with the HE4
fragment). The CIHDpa vector, provided by Dr. Say Kong Ng of the
National University of Singapore, utilizes destabilizing sequences
on a selection marker for improved recombinant protein productivity
in CHO-DB44 cells, as described in Ng et al., Metab Eng 9:304
(2007)), incorporated herein by reference. Briefly described, the
CIHDpa expression vector contains a standard cytomegalovirus (CMV)
promoter upstream of the cDNA insertion site. A herpes simplex
virus thymidine kinase (HSV-th) promoter is positioned upstream of
a dihydrofolate reductase (dhfr) gene, which serves as a selective
marker for successful transfectants (i.e., cells deficient in dhfr
require hypoxanthine and thymidine supplements to survive).
Immediately downstream of the dhfr marker is a murine ornithine
decarboxylase PEST (MODC PEST) region and a series of AU-rich
elements (ARE), which are expressed as fusion components at the
carboxy terminal end of the dhfr protein. The MODC PEST peptide
serves as a degradation signal leading to instability of the marker
protein. The ARE region serves to destabilize the mRNA encoding the
marker protein. With the dhfr marker destabilized at the
transcription and translation levels, only transfected cells that
have highly efficient production of the plasmid-encoded proteins
will survive, thus leading to improved productivity of the
recombinant HE4 protein after selection.
[0089] The resulting expression vector containing HE4, designated
"HE4-CIHDpa," was confirmed by restriction enzyme analysis, and the
inserted HE4 cDNA was also confirmed by sequencing the complete
cDNA insert.
[0090] Transfection of HE4-CIHDpa Plasmid into Chinese Hamster
Ovary (CHO) Cells
[0091] Dihydrofolate reductase deficient (DHFR-) Chinese Hamster
Ovary (CHO-DG44) cells were grown in serum-free CHO medium
(Hyclone, Logan, Utah; SH30333) at 37.degree. C., 5% CO.sub.2. 0.5
million or 1 million CHO-DG44 cells were seeded in each well of a
24-well plate with 500 .mu.I serum-free medium per well and were
incubated overnight at the conditions described above. 1 .mu.g or 5
.mu.g of endo-toxin free HE4-CIHDpa plasmid was combined with 2
.mu.l Lipofectamine.TM. 2000 (Invitrogen) in 100 .mu.l Opti-MEM
serum-free medium. After incubating for 20 minutes, the
DNA-Lipofectamine.TM. complexes were added into each well
containing CHO-DG44 cells. The cells were then transferred into
hypoxanthine and thymidine ("HT"). HT-supplemented HyQ PF-CHO
medium and allowed to recover for 2 days before transferring to HyQ
PF-CHO medium without the HT supplement to select for
transfectants. After 28 days in the selective medium, 1 million
cells transfected with 1 .mu.g plasmid grew and recovered with a
cell viability >95%.
[0092] Production and Purification of HE4 Protein by Transfected
CHO Cells
[0093] HE4 protein production in culture supernatants from CHO
cells transfected with HE4-CIHDpa plasmid was evaluated by using a
commercially available kit, marketed by Fujirebio Diagnostics,
Inc., which is based on a double determinant (Sandwich) ELISA
assay, as described in Hellstrom I., et al., Cancer Research
63:3695-3700 (2003), incorporated herein by reference.
[0094] As described in Hellstrom et al. (2003), a monoclonal Ab 3D8
(specific for one epitope on HE4) was coated overnight onto the
wells of an assay plate, after which 100 .mu.l supernatant from the
CHO culture transfected with HE4-CIHDpa plasmid was added to the
cells and incubated for 1 hour. The assay plate was then incubated
with a biotinylated second anti-HE4 monoclonal Ab, 2H5 (specific
for a different HE4 epitope) for 1 hour.
[0095] TMB (3,3',5,5'-tetramethylbenzidine), a chromogenic
substrate for Peroxidase (KPL, Gaithersburg, Md.) was added and
permitted to incubate for 15 minutes. Optical density (OD) readings
were made at 450 nm. The OD450 from CHO supernatant was 3.1, as
compared with 0.078 for medium alone, indicating that HE4 protein
was highly expressed by the CHO cells and secreted into the culture
medium.
[0096] Purification of HE4 Recombinant Protein by Affinity
Chromatography
[0097] The recombinant HE4 protein was purified from high producing
lines of CHO cells transfected with HE4-CIHDpa plasmid by affinity
chromatography as follows.
[0098] Anti-HE4 monoclonal Abs 3D8 and 2H5 were coupled to
6-aminohexanoic acid N-hydroxysuccinimide ester-activated-Sepharose
4B (Sigma, St. Louis, Mo.; #A9019). Briefly described, 0.5 g of
powder was swollen in 1 mM HCl for 15 minutes. The beads were
drained and resuspended in 3 mg anti-HE4 monoclonal Ab in 1 ml of
0.5 M NaCl/0.1 M NaHCO.sub.3 (pH 8.3) solution. The column was
stored at 4.degree. C. overnight. The next day, the resin was
blocked with 1 M Tris-HCl for 2 hours. The unbound anti-HE4
monoclonal Ab was removed by washing the resin with
phosphate-buffered saline (PBS). Supernatant from large scale
cultures of CHO cells transfected with HE4-CIHDpa plasmid was
collected and the pH was adjusted with NaHCO.sub.3 to pH 8.0. The
adjusted cell supernatant was applied to the column, and bound HE4
protein was eluted with 0.1 M glycine-HCl (pH 2.7) and neutralized
with 200 .mu.l 2M Tris-HCl. The eluted HE4 protein was concentrated
with a 10 kD centrifugal filter tube (Millipore, Billerica, Mass.).
The column purified HE4 recombinant protein was assayed by Sandwich
ELISA, which was performed as described above. The results of the
ELISA assays are shown below in TABLE 1.
TABLE-US-00001 TABLE 1 Presence of HE4 protein as determined by
Sandwich ELISA. Samples Dilution factor OD450 Non-purified CHO
supernatant undiluted 2.832 (i.e. before column purification) CHO
medium control undiluted 0.110 Column-Purified HE4 undiluted 3.432
protein (0.1 .mu.g)
[0099] The column purified human recombinant HE4 protein obtained
from CHO cells was obtained as described above and pooled for use
in developing an ELISA assay to detect anti-HE4 antibody in patient
samples, as described in EXAMPLE 2. The recombinant human HE4
protein was also tested in ELISA assays for binding to mouse
anti-HE4 antibodies, and it was determined that the mouse anti-HE4
antibodies recognized both native and recombinant human HE4 protein
(data not shown).
[0100] Conclusion:
[0101] These, results demonstrate the successful cloning,
expression, and purification of recombinant human HE4 protein from
CHO cells.
Example 2
[0102] This Example describes the successful development of an
ELISA assay capable of detecting naturally occurring human anti-HE4
antibodies in human serum.
[0103] Methods:
[0104] Development of an ELISA Assay for Detecting Anti-HE4
Antibodies:
[0105] An ELISA assay was developed for detecting/measuring the
amount of anti-HE4 antibodies in serum as follows. 0.6 .mu.g/mL of
purified recombinant human HE4 protein (produced as described in
EXAMPLE 1), was coated overnight onto the wells of an ELISA assay
plate. Matching wells were left uncoated as controls for
background. After blocking for 2 hours with 3% BSA/PBS, human sera
or plasma at dilutions of 1:20 and 1:40 (obtained as described
below) were added to all of the wells on the ELISA assay plate. All
dilutions of both samples and reagents were performed in 3%
BSA/PBS.
[0106] After incubating with HRP-conjugated mouse anti-human IgG
antibody and TMB substrate, the assay plate was scanned with a
Dynatech MR 5000 plate reader at 450 nm. The background control
(stickiness due to the innate nature of IgG to stick
non-specifically to the control wells of the ELISA assay plate) was
measured as OD450 in the parallel control wells (to which no
antigen had been attached), and the background control values were
subtracted from the reading in the coated wells to give the final
IgG level.
[0107] Plasma Samples Obtained from Ovarian Cancer Patients
[0108] A pilot study was performed in which plasma samples were
obtained from ten ovarian cancer patients, most of whom had
advanced (stage III-IV) disease, and one healthy female subject,
and tested in the anti-HE4 ELISA assay in order to test for the
presence of naturally occurring anti-HE4 antibodies. The human
plasma samples were added to each well of the ELISA plate at 1:20
and 1:40 dilutions and were incubated at room temperature for 1
hour. The wells were washed with PBS-Tween 20, and then 1:1000
diluted HRP-conjugated mouse anti-human IgG antibody (Invitrogen,
Carlsbad, Calif.) was added to each well and incubated for 1 hour
at room temperature. After washing the plate again with PBS-Tween
20, SureBlue.TM. TMB Microwell Peroxidase Substrate (KPL) was added
to each well and incubated for 15 minutes at room temperature,
after which the interaction was terminated by adding the TMB stop
solution (KPL). Optical density (OD) at 450 nanometers was measured
with a DynaTech MR 5000 plate reader (DynaTech Laboratories,
Inc.).
[0109] Results:
[0110] The results of the ELISA assay testing for the
presence/amount of anti-HE4 antibodies from the human sera samples
are presented in FIG. 1. As shown in FIG. 1, one of the ten
patients with ovarian carcinoma (subject He213) had an OD450
.gtoreq.1.0 for both dilutions, thus indicating the presence of
anti-HE4 antibodies. It is noted that the plasma from patient He213
(with plasma that was positive for anti-HE4 abs), was found to be
negative for the presence of HE4 antigen using the assay to detect
the presence of HE4 antigen as described in U.S. Pat. No.
7,270,960, incorporated herein by reference (data not shown).
However, cultured tumor cells derived from the tumor of patient
He213 were shown to express the HE4 antigen (data not shown).
[0111] Conclusion:
[0112] The results described in this example demonstrate the
successful development of an ELISA assay capable of detecting
naturally occurring human anti-HE4 antibodies in human serum. The
results also demonstrate the presence of anti-HE4 antibodies in a
subject with ovarian cancer. Based on our previous study on
antibodies to mesothelian, (see Hellstrom I. et al., Cancer
Epidemiol Biomarkers Prev. 17(6):1520-1526 (2008)), it is expected
that the presence of, or an increase in titer of antibodies to HE4
identified in ovarian cancer patients that are undergoing, or
having undergone treatment (via surgery, chemotherapy, radiation
and/or immunotherapy), either alone, or in combination with a
finding of a decreased amount of HE4 antigen (as compared to the
baseline amount at the start of the treatment), or no circulating
HE4 antigen, will indicate that the cancer therapy has been
effective in damaging the tumor. It is further expected that a
decrease in the titer of anti-HE4 antibodies, or no detectable
anti-HE4 antibodies to HE4 in ovarian cancer patients undergoing
treatment, either alone, or in combination with increased amount of
circulating HE4 antigen (as compared to the baseline amount at the
start of treatment), will indicate that the cancer therapy has been
ineffective in damaging the tumor, and/or indicative of a
relapse.
Example 3
[0113] This Example describes a study in which the ELISA assay
developed for detecting the presence/amount of antibodies to HE4
(described in EXAMPLE 2) was used to titrate the amount of anti-HE4
antibody present in serum from 3 patients with ovarian carcinoma,
and 20 healthy female control subjects.
[0114] Methods:
[0115] Serum Samples Obtained from Ovarian Cancer Patients
[0116] As shown in TABLE 2 below, serum samples from three patients
with early stage (I/II) ovarian carcinoma and 20 healthy human
female subjects were obtained at the Fred Hutchinson Cancer
Research Center (Seattle, Wash.), 4 of which are shown in Table 2
below. Sera were drawn from the subjects and assayed using the
methods as described in Example 2.
[0117] Results:
[0118] The sera obtained as described above were assayed at 1:20
and 1:40 dilutions using the ELISA method described in EXAMPLE 2.
The results are summarized in TABLE 2 below.
TABLE-US-00002 TABLE 2 Results of HE4 antibody analysis of serum
from three ovarian Cancer patients, and 4 healthy controls using
the ELISA assay to detect/measure HE4 antibodies. OD450 > 0.500
Clinical (at least in the Patient Code Disease/Condition 1:20
dilution 100C17 Ovarian Carcinoma + 206241 Ovarian Carcinoma +
208448 Ovarian Carcinoma + 1 healthy female control - 2 healthy
female control - 3 healthy female control - 4 healthy female
control - 5-24 (total of 24 Healthy female control 3 positive/21
negative healthy female (of 24 total tested) control subjects)
[0119] As summarized in TABLE 2, all three serum samples from
patients with ovarian carcinoma, and three of the 24 serum samples
from healthy female subjects had an OD450 .gtoreq.0.5 for the 1:20
dilution. As shown in TABLE 2, all of the three patients with
ovarian cancer had anti-HE4 antibodies in their serum, as indicated
by an OD450 .gtoreq.0.5 at the 1:20 dilution. These results are
consistent with the results described in Example 2, and further
demonstrate the successful development of an ELISA assay capable of
detecting naturally occurring human anti-HE4 antibodies in human
serum. The results also demonstrate the presence of anti-HE4
antibodies in a subjects with both early and later stage ovarian
cancer.
Example 4
[0120] Reactivity with HE4 Fragments Displayed as Phage Fusion
Proteins
[0121] The epitope specificity of the human Anti HE4 antibodies was
determined by testing the reactivity of human samples that
displayed anti HE4 reactivity towards HE4 fragments expressed as
fusion proteins with phage coat protein pVIII in a phage ELISA.
[0122] Cloning of HE4 fragments in f88-4 cDNA, prepared from mRNA
isolated from OvCar-3 cells, served as template for PCR
amplification of the gene parts coding for the HE4 domains for
cloning in the phage display vector f88-4. PCR primer pairs, listed
in Table 3, were constructed for amplification of the coding
regions indicated in Figure x. In the 5'-ends were restriction
sites for HindIII and PstI inserted for cloning in fusion with the
pVIII signal peptide and the pVIII mature coat protein.
TABLE-US-00003 TABLE 3 PCR primers used for amplification of HE4
fragments Primer Sequence 5' to 3' WAP W1F1 TGCTAAGCTTTGCC
GAGAAGACTGGCGTGTGCCC N-WAP W1F2 TGCTAAGCTTTGCC AGCGAATGCGCCGACAACC
N-WAP W1F3 TGCTAAGCTTTGCC GACCAGAACTGCACGCAAG N-WAP W1R1
CCTTCTGCAGG ATCATTGGGCAGAGAGCAG N-WAP W1R2 CCTTCTGCAGG
GTCCGAGACGCACTCTTGC N-WAP W1R3 CCTTCTGCAGG GCTGCAGCACTTGAGGTTG
N-WAP W2F1 TGCTAAGCTTTGCC AAGGAGGGTTCCTGCCCCCA C-WAP W2F2
TGCTAAGCTTTGCC AGCCAGTGTCCTGGCCAG C-WAP W2F3 TGCTAAGCTTTGCC
CAGCTCGGCCTCTGTCGGGAC C-WAP W2R1 CCTTCTGCAGG GAAATTGGGAGTGACACAGGA
C-WAP W2R2 CCTTCTGCAGG GTCCACCTGGCACTGGTCC C-WAP W2R3 CCTTCTGCAGG
ATTGCGGCAGCATTTCATCTG C-WAP
[0123] The HE4 fragments were separately amplified from 0.5 .mu.l
of cDNA in a reaction mixture containing 1 .mu.M of each forward
and reverse primer, 75 mM Tris-HCl (pH 8.8 at 25.degree. C.), 20 mM
(NH.sub.4).sub.2SO.sub.4, 0.1% (v/v) Tween 20, 2 mM MgCl.sub.2,
0.02 u/.mu.l Taq-polymerase (Abgene, Surrey, UK) and 0.1 mM of each
deoxynucleotide in a final volume of 25 .mu.I with the following
temperature cycle repeated 30 times: 30 seconds incubations at
95.degree. C., 50.degree. C. and 72.degree. C.
PCR products and f88-4, digested with HindIII and PstI, were
ligated together and transfected into E. coli JM109 where after
clones were selected on LB plates with tetracycline. Two clones of
each construct were amplified in E. coli JM109 and double-stranded
DNA was prepared for DNA sequencing. DNA sequencing was performed
using the Big dye terminator v1.1 cycle sequencing kit and a f88-4
vector specific primer. Sequencing reactions were sent to CyberGene
AB (Huddinge, Sweden) for analysis. Sequence raw data was analyzed
using the free software Chromas version 1.45 (Technelysium Pty
Ltd., Australia). Nucleotide sequencing verified insertion in frame
with the leader peptide and the mature phage coat protein pVIII.
The HE4 inserts demonstrated identity to the expected HE4 fragment
sequences (accession number AY212888). The positions of the HE4
fragments expressed as fusion proteins with coat protein pVIII
using the primers shown in Table 1 is shown in FIG. 2. The amino
acid numbers refer to positions is the HE4 amino acid sequence set
forth in SEQ ID NO: 2.
Phage ELISA
[0124] Sequence verified phage clones were amplified, purified,
concentrated with PEG/NaCl and were diluted in PBS for use as
coating antigen in the direct phage ELISA assay, or alternatively
in 1% BSA in PBS as antigen in the sandwich phage ELISA.
[0125] In the direct phage ELISA the HE4 fragment phages were
diluted in carbonate buffer pH 9.2 and coated in microliter wells.
Binding of the human anti HE4 ab's was determined after dilution of
the patient serum in PBS-1% BSA and incubation in HE4 phage coated
plates. The bound hIg was determined by incubation with HRP Anti
hIg Ab.
[0126] In the sandwich HE4 phage ELISA the patient samples were
diluted with PBS-1% BSA and incubated in microtiter plates coated
with anti-Human IgG for adsorption of the hIgG. The coated plates
were incubated with the different HE4 pVIII phage particles in a
volume of 100 .mu.l/well were added. After two hours incubation,
wells were washed and a rabbit anti-M13 antibody (established
in-house) was added. After incubation and washing, a HRP labelled
swine anti rabbit antibody (Dako) was added. After the final wash
TMB substrate was added and the plate was measured at 620 nm after
5 minute incubation. wt M-13 phage was used as negative control n
both ELISA assays.
[0127] While the preferred embodiment of the invention has been
illustrated and described, it will be appreciated that various
changes can be made therein without departing from the spirit and
scope of the invention.
SEQUENCE LISTING
TABLE-US-00004 [0128] SEQ ID NO: 1: HE4 cDNA (human). 486 bp, CDS:
38-418 GenBank:AY212888.1 Homo sapiens HE4 protein (WFDC2) mRNA,
complete cds
TGAGAGAAAGCGGCCGCACCCCGCCCGGCATAGCACCATGCCTGCTTGTCGCCTAGGCCCGCTAGCCGCC
GCCCTCCTCCTCAGCCTGCTGCTGTTCGGCTTCACCCTAGTCTCAGGCACAGGAGCAGAGAAGACTGGCG
TGTGCCCCGAGCTCCAGGCTGACCAGAACTGCACGCAAGAGTGCGTCTCGGACAGCGAATGCGCCGACAA
CCTCAAGTGCTGCAGCGCGGGCTGTGCCACCTTCTGCTCTCTGCCCAATGATAAGGAGGGTTCCTGCCCC
CAGGTGAACATTAACTTTCCCCAGCTCGGCCTCTGTCGGGACCAGTGCCAGGTGGACAGCCAGTGTCCTG
GCCAGATGAAATGCTGCCGCAATGGCTGTGGGAAGGTGTCCTGTGTCACTCCCAATTTCTGAGCTCCGGC
CACCACCAGGCTGAGCAGTGAAGATAGAAAGTTTCTGCCTGGCCCTGCAGCGTGTTACAGCCCACC
SEQ ID NO: 2: HE4 protein (human) (124 aa) GenBank:AAO52683.1 HE4
protein [Homo sapiens]
MPACRLGPLAAALLLSLLLFGFTLVSGTGAEKTGVCPELQADQNCTQECVSDSECADNLKCCSAGCATFCSL
PNDKEGSCPQVNINFPQLGLCRDQCQVDSQCPGQMKCCRNGCGKVSCVTPNF SEQ ID NO: 3:
(forward primer) 5'-AAAAACCGGTATGCCTGCTTGTCGCCTAGG-3 SEQ ID NO: 4
(reverse primer) 5'-AAAACCTGCAGGTCAGAAATTGGGAGTGACAC-3'
Sequence CWU 1
1
261486DNAHomo sapiens 1tgagagaaag cggccgcacc ccgcccggca tagcaccatg
cctgcttgtc gcctaggccc 60gctagccgcc gccctcctcc tcagcctgct gctgttcggc
ttcaccctag tctcaggcac 120aggagcagag aagactggcg tgtgccccga
gctccaggct gaccagaact gcacgcaaga 180gtgcgtctcg gacagcgaat
gcgccgacaa cctcaagtgc tgcagcgcgg gctgtgccac 240cttctgctct
ctgcccaatg ataaggaggg ttcctgcccc caggtgaaca ttaactttcc
300ccagctcggc ctctgtcggg accagtgcca ggtggacagc cagtgtcctg
gccagatgaa 360atgctgccgc aatggctgtg ggaaggtgtc ctgtgtcact
cccaatttct gagctccggc 420caccaccagg ctgagcagtg aagatagaaa
gtttctgcct ggccctgcag cgtgttacag 480cccacc 4862124PRTHomo sapiens
2Met Pro Ala Cys Arg Leu Gly Pro Leu Ala Ala Ala Leu Leu Leu Ser 1
5 10 15 Leu Leu Leu Phe Gly Phe Thr Leu Val Ser Gly Thr Gly Ala Glu
Lys 20 25 30 Thr Gly Val Cys Pro Glu Leu Gln Ala Asp Gln Asn Cys
Thr Gln Glu 35 40 45 Cys Val Ser Asp Ser Glu Cys Ala Asp Asn Leu
Lys Cys Cys Ser Ala 50 55 60 Gly Cys Ala Thr Phe Cys Ser Leu Pro
Asn Asp Lys Glu Gly Ser Cys 65 70 75 80 Pro Gln Val Asn Ile Asn Phe
Pro Gln Leu Gly Leu Cys Arg Asp Gln 85 90 95 Cys Gln Val Asp Ser
Gln Cys Pro Gly Gln Met Lys Cys Cys Arg Asn 100 105 110 Gly Cys Gly
Lys Val Ser Cys Val Thr Pro Asn Phe 115 120 330DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Forward sense
primer 3aaaaaccggt atgcctgctt gtcgcctagg 30432DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Reverse
antisense primer 4aaaacctgca ggtcagaaat tgggagtgac ac 32545PRTHomo
sapiens 5Glu Lys Thr Gly Val Cys Pro Glu Leu Gln Ala Asp Gln Asn
Cys Thr 1 5 10 15 Gln Glu Cys Val Ser Asp Ser Glu Cys Ala Asp Asn
Leu Lys Cys Cys 20 25 30 Ser Ala Gly Cys Ala Thr Phe Cys Ser Leu
Pro Asn Asp 35 40 45 649PRTHomo sapiens 6Lys Glu Gly Ser Cys Pro
Gln Val Asn Ile Asn Phe Pro Gln Leu Gly 1 5 10 15 Leu Cys Arg Asp
Gln Cys Gln Val Asp Ser Gln Cys Pro Gly Gln Met 20 25 30 Lys Cys
Cys Arg Asn Gly Cys Gly Lys Val Ser Cys Val Thr Pro Asn 35 40 45
Phe 722PRTHomo sapiens 7Glu Lys Thr Gly Val Cys Pro Glu Leu Gln Ala
Asp Gln Asn Cys Thr 1 5 10 15 Gln Glu Cys Val Ser Asp 20 822PRTHomo
sapiens 8Asp Gln Asn Cys Thr Gln Glu Cys Val Ser Asp Ser Glu Cys
Ala Asp 1 5 10 15 Asn Leu Lys Cys Cys Ser 20 923PRTHomo sapiens
9Ser Glu Cys Ala Asp Asn Leu Lys Cys Cys Ser Ala Gly Cys Ala Thr 1
5 10 15 Phe Cys Ser Leu Pro Asn Asp 20 1025PRTHomo sapiens 10Lys
Glu Gly Ser Cys Pro Gln Val Asn Ile Asn Phe Pro Gln Leu Gly 1 5 10
15 Leu Cys Arg Asp Gln Cys Gln Val Asp 20 25 1124PRTHomo sapiens
11Gln Leu Gly Leu Cys Arg Asp Gln Cys Gln Val Asp Ser Gln Cys Pro 1
5 10 15 Gly Gln Met Lys Cys Cys Arg Asn 20 1214PRTHomo sapiens
12Gln Leu Gly Leu Cys Arg Asp Gln Cys Gln Val Asp Ser Gln 1 5 10
1324PRTHomo sapiens 13Ser Gln Cys Pro Gly Gln Met Lys Cys Cys Arg
Asn Gly Cys Gly Lys 1 5 10 15 Val Ser Cys Val Thr Pro Asn Phe 20
1448PRTHomo sapiens 14Ser Glu Cys Ala Asp Asn Leu Lys Cys Cys Ser
Ala Gly Cys Ala Thr 1 5 10 15 Phe Cys Ser Leu Pro Asn Asp Lys Glu
Gly Ser Cys Pro Gln Val Asn 20 25 30 Ile Asn Phe Pro Gln Leu Gly
Leu Cys Arg Asp Gln Cys Gln Val Asp 35 40 45 1534DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Forward sense
primer 15tgctaagctt tgccgagaag actggcgtgt gccc 341633DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Forward sense
primer 16tgctaagctt tgccagcgaa tgcgccgaca acc 331733DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Forward sense
primer 17tgctaagctt tgccgaccag aactgcacgc aag 331830DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Reverse sense
primer 18ccttctgcag gatcattggg cagagagcag 301930DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Reverse sense
primer 19ccttctgcag ggtccgagac gcactcttgc 302030DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Reverse sense
primer 20ccttctgcag ggctgcagca cttgaggttg 302134DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Forward sense
primer 21tgctaagctt tgccaaggag ggttcctgcc ccca 342232DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Forward sense
primer 22tgctaagctt tgccagccag tgtcctggcc ag 322335DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Forward sense
primer 23tgctaagctt tgcccagctc ggcctctgtc gggac 352432DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Reverse sense
primer 24ccttctgcag ggaaattggg agtgacacag ga 322530DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Reverse sense
primer 25ccttctgcag ggtccacctg gcactggtcc 302632DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Reverse sense
primer 26ccttctgcag gattgcggca gcatttcatc tg 32
* * * * *